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Copper binding modulates the platination of human copper chaperone Atox1 by antitumor trans-platinum complexes† Zhaoyong Xi,a Wei Guo,b Changlin Tian,*c Fuyi Wang*b and Yangzhong Liu*a The transport system of platinum-based anticancer agents is crucial for drug sensitivity. Increasing evidence indicates that the copper transport system is also involved in the cellular influx and efflux of platinum drugs. The copper chaperone Atox1 has been shown to bind to cisplatin in vitro and in cells. Previous results reveal that copper binding promotes the reaction between Atox1 and cisplatin. Here, we have performed detailed solution NMR and ESI-MS experiments to investigate the effect of Cu(I) binding on the reactions of Atox1 with two antitumor active trans-platinum agents, trans-EE and trans-PtTz. Results indicate that, similar to the reaction of cisplatin, copper coordination also enhances the platination of Atox1 by two trans-platinum complexes, and platinum binds to the copper coordinating residues. However, copper binding promotes the trans-platinum transfer from Atox1 to dithiothreitol

Received 15th November 2013, Accepted 6th January 2014

(DTT). This result is in contrast to the reaction of Atox1 with cisplatin, in which the presence of copper

DOI: 10.1039/c3mt00338h

platinum complexes than with cisplatin, however, less protein aggregation is observed in the reaction of trans-platinum complexes. These results indicate that the roles of Atox1 in the regulation of cellular

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trafficking of platinum drugs are dependent on the coordination configurations.

largely suppresses the platination of DTT. Additionally, both apo- and CuI-Atox1 react faster with trans-

Introduction Cisplatin is an efficient anticancer agent in the treatment of a variety of solid tumors; the platinum binding leads to the DNA damage and induces apoptosis.1 However, the clinical use of platinum-based drugs is seriously limited due to intrinsic and acquired resistance.2 Since the discovery of the antitumor properties of cisplatin and its analogues, the presence of a cis configuration with two leaving groups has long been considered to be essential for the anticancer activity. In recent years, a number of trans-platinum compounds have shown promising antitumor activities, such as trans-[PtCl2{E-HNQC(OCH3)CH3}2] (trans-EE) and trans-[PtCl2(NH3)(thiazole)] (trans-PtTz).3,4

a

CAS High Magnetic Field Laboratory, CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry & Collaborative Innovation Center of Suzhou Nano Science and Technology, University of Science and Technology of China, Hefei, Anhui, China. E-mail: [email protected]; Tel: +86-551-63600874 b Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing, China. E-mail: [email protected] c Hefei National Laboratory of Microscale Physical Sciences, School of Life Science, University of Science and Technology of China, Hefei, Anhui, China. E-mail: [email protected] † Electronic supplementary information (ESI) available: ESI-MS spectra and SDS-PAGE. See DOI: 10.1039/c3mt00338h

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In particular, trans-EE exhibits higher cytotoxicity than its cis congener, and also active to several cisplatin-resistant tumor cell lines.3 These findings imply an alternative strategy for platinum drug design, especially for the cisplatin resistant cells. Protein interactions have been proved to play important roles in drug resistance.2,5 Increasing evidence indicates that the copper transport proteins, Ctr1 and ATPases mediate the influx and efflux of platinum drugs, respectively.6,7 Human copper chaperone Atox1, which delivers Cu(I) ions to ATPase, is believed to be involved in cisplatin resistance. X-ray crystallography and NMR measurements demonstrated that cisplatin binds to Atox1 at the copper coordinating residues.8,9 It has been also reported that Atox1 could regulate the cellular accumulation of cisplatin and transfer cisplatin to the metal binding domains of ATP7A and ATP7B; and high level of Atox1 was found in cisplatinresistant cells.10–14 Recently, we have found that the copper coordination enhances the reactivity of Atox1 to cisplatin, which could affect the copper protein associated drug resistance.15 Due to the different coordination chemistry, trans-platinum complexes exhibit significantly different kinetics and binding modes from cisplatin in DNA binding.4,16 These divergences are considered as the origins for the different drug activities of various platinum complexes. Due to the significance and the diversity of protein reactions in drug resistance, the direct interactions of proteins with platinum complexes could also

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HPLC-ESI-MS

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Scheme 1

Structures of platinum complexes.

contribute to the different antitumor activities. To understand the effect of the configurations of platinum complexes on the reactions of Atox1, we performed the reactions on two antitumor active trans-platinum complexes (trans-EE and trans-PtTz, see Scheme 1). Because of the cross resistance between copper and cisplatin,17,18 we also studied the effect of copper binding on the platination of Atox1. The reaction rates were measured through the 2D 1H–15N HSQC spectroscopy with the 15N isotope labeled trans-platinum complexes. The products were characterized using tandem mass spectroscopy. Results showed the different roles of Atox1 and copper coordination in the reactions of transplatinum complexes in comparison with cisplatin.

Experimental Synthesis of platinum drugs The 15N-labeled platinum complexes, trans-[PtCl2{E-H15NQC(OCH3)(CH3)}2] and trans-[PtCl2(15NH3)(thiazole)] were synthesized according to literature methods.19,20 Protein expression and purification The gene sequence for the Atox1 expression was amplified via PCR from a human cDNA library. Atox1 in pST-SG1 vector was transformed into BL21(DE3) Gold cells. The cells were grown in LB medium at 37 1C. When OD600 reached 0.8, the culture was induced with 0.8 mM IPTG for 4 h. Cells were harvested by centrifugation at 4000 rpm at 18 1C for 20 min. The protein was purified using Ni2+ affinity chromatography. After TEV protease digestion to remove the (His)6 tag, the protein was further purified through size-exclusion chromatography using a Superdex 75 16/60 column (GE Healthcare). NMR spectroscopy Protein samples were prepared in a buffer of 50 mM sodium phosphate and 5-fold DTT at pH 7.0. For the preparation of CuI-Atox1, an equimolar amount of Cu(I) complex [Cu(CH3CN)4]+ was added to apo-Atox1 in the buffer containing DTT. The NMR samples were prepared by mixing protein solutions with equimolar lyophilized platinum complexes to a final concentration of 0.8 mM. 1 H–15N HSQC spectra were recorded immediately to monitor the reactions. All NMR experiments were carried out on a 700 MHz Varian Inova spectrometer at 25 1C. The spectral widths were set as 10 ppm (1H) and 20 ppm (15N, centered at 92 ppm) for the trans-EE reactions, and 8 ppm (1H) and 20 ppm (15N, centered at 65 ppm) for the trans-PtTz reactions. The pulse sequence was optimized with a delay 1/(4JNH) of 3.42 ms (trans-PtTz) or 3.21 ms (trans-EE). 15N chemical shifts of platinum complexes were referenced to 15NH4Cl.

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For time-dependent measurements, samples of 0.2 mM Atox1 were incubated with equimolar platinum complexes at 25 1C for different times. The resulting mixtures were directly analyzed by HPLC-ESI-MS. For trypsin digestion, Atox1 samples were treated with equimolar platinum complexes at 25 1C for 4 h. The unbound platinum complexes were removed on HPLC with a Zorbax eclipse XDB-C8 column. Then, trypsin (from Promega) was added to the purified protein solution in a molar ratio of 1 : 40 (trypsin : Atox1). The digestion mixture was incubated for 6 h at 37 1C prior to HPLC-ESI-MS analysis. ESI-MS spectra were recorded on a Micromass Q-TOF mass spectrometer (Waters) coupled to a Waters CapLC HPLC system. Pt-Atox1 adducts were separated on a symmetry-C8 column (Waters), while the tryptic digests of Pt-Atox1 adducts were separated on a symmetry-C18 column (Waters). Linear gradients were used (mobile phases: A: H2O/CH3CN/HCOOH (95 : 4.9 : 0.1); B: H2O/CH3CN/HCOOH (4.9 : 95 : 0.1)). The eluents were directly delivered into the mass spectrometer through the ESI probe. The MS spectra were obtained in the range of 500–1800 m/z at a capillary temperature of 140 1C with the spray voltage of 3.30 kV and the cone voltage of 35 V. The collision energy was set to 10 V. Collision-induced dissociation MS/MS spectra were acquired in the range of 100–2000 m/z, and the relative collision energy was set to 15–20 eV.

Results NMR studies on the reaction between Atox1 and trans-EE The reaction of apo-Atox1 with 15N-labeled trans-EE was measured using 2D 1H–15N HSQC NMR spectroscopy. The signals of dichloro trans-EE (1, 89.2/7.43 ppm) and its hydrolysis species (2, 92.9/7.51 ppm) are in accordance with our previous report21 (Fig. 1A). The binding of trans-EE to Atox1 generated a new signal (peak 3) at 97.6/7.81 ppm. In addition to the platinated Atox1 adduct, several trans-EE/DTT adducts were also detected based on the control experiments. During the formation of the platinated Atox1, the signals of trans-EE decreased quickly and disappeared completely after 33 min. This reaction is considerably faster than the reaction of cisplatin with apo-Atox1, in which the half-life time is about 170 min.15 The reaction of CuI-Atox1 showed that the signals of trans-EE disappeared more quickly in the reaction of holo-Atox1 than that of apo-Atox1 (Fig. 1 and Fig. S1, ESI†). Meanwhile, more trans-EE–Atox1 adducts and less trans-EE–DTT adducts were formed in the reaction of CuI-Atox1 in the early stage (Bhalfhour) (Fig. 2). This result indicates that, similar to the reaction of cisplatin,15 the copper coordination also enhances the reactivity of Atox1 towards trans-EE and suppresses the platination of DTT. After 26 min of reaction, trans-EE–Atox1 significantly decreased the intensity (B59% decreased during 8 h of reaction) in the reaction of CuI-Atox1, while trans-EE–DTT increased to about twice the amount. As no free trans-EE was present during the period, these intensity changes indicate the platinum transfer from Atox1 to DTT (Fig. 2). In contrast, in the

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observation clearly indicates that the copper coordination promotes the platinum transfer from Atox1 to DTT. This result is different from the reaction of cisplatin, in which the copper coordination significantly suppresses the platination of DTT throughout the reaction.15

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NMR studies on the reaction of Atox1 with trans-PtTz

Fig. 1 1H–15N HSQC spectra of 15N labeled trans-EE at 25 1C in the reaction with Atox1 at different times. (A) apo-Atox1; (B) CuI-Atox1. The cross-peaks in the spectra were assigned to trans-EE (1); monochloro trans-EE (2); the adduct of trans-EE with Atox1 (3) and the adducts of trans-EE with DTT (#).

To confirm the different effects of copper binding on the platinum transfer are dependent on the configuration of platinum complexes, the reaction of Atox1 was also performed on another antitumor active trans-platinum complex, trans-PtTz. Even no free trans-PtTz or its hydrolysate signals were observed in the first 1H–15N HSQC spectrum at 9 min of reaction with apo-Atox1 (Fig. 3A), indicating that trans-PtTz is much more reactive than cisplatin or trans-EE. Meanwhile, three major cross-peaks appeared, including an intense cross-peak at 64.8/3.91 (4) ppm from the trans-PtTz–Atox1 adduct and the other two peaks from DTT adducts. With the progress of the reaction, peak 4 decreased in its intensity slowly, while the amount of DTT adducts increased. This observation indicates the platinum transfer from Atox1 to DTT since no free trans-PtTz existed during this process. In the reaction of CuI-Atox1, the same resonance of the transPtTz–Atox1 adduct (4) was observed after 9 min of incubation, however, with a much higher intensity (Fig. 3B). This result confirmed that copper coordination promotes the platination of Atox1 by trans-PtTz as well as cisplatin and trans-EE. Surprisingly, this signal decreased rapidly and became undetectable within 1.5 h. Meanwhile, trans-PtTz/DTT adducts were observed with increasing intensities. These results clearly show that the copper coordination promotes the transfer of trans-PtTz from Atox1 to DTT.

Fig. 2 The time dependent concentrations of platination adducts in the reaction of trans-EE with Atox1 detected in the NMR spectra. The color of columns denotes the reaction of apo-Atox1 (white) or CuI-Atox1 (gray). (A) The trans-EE–Atox1 adduct (peak 3 in the 1H–15N HSQC spectra); (B) the trans-EE–DTT adducts.

reaction of apo-Atox1, the amount of platination adducts almost did not change during this period (from 33 min to 8 h). This

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Fig. 3 1H–15N HSQC spectra of 15N labeled trans-PtTz at 25 1C in the reaction with Atox1 at different times. (A) apo-Atox1; (B) CuI-Atox1. The cross-peaks in the spectra were assigned to the adduct of trans-PtTz with Atox1 (4) and the adducts of trans-PtTz with DTT (#).

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Therefore, copper coordination has the same effect on the platinum transfer of two trans-platinum complexes, but in contrast to that of cisplatin.

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ESI-MS studies on the species of Atox1 with trans-EE and trans-PtTz The platinated products of Atox1 were characterized with timedependent ESI-MS (Fig. 4 and 5). Different from the reaction of cisplatin, the carrier ligands (iminoether, Ime) remained coordinated to platinum throughout the reaction of trans-EE (Fig. 4A). The platinated Atox1 adducts were clearly observed after 10 min of reaction, including [Atox1 + Pt(Ime)2 + 6H]8+ (a1, m/z 1002.51) and [Atox1 + Pt(Ime)2 + DTT + 6H]8+ (a2, m/z 1021.75) (see Table 1 for the peak assignments). Bis-platinated adducts (a3, a4 and a5) were observed after 1 h of reaction. As a1 is always the dominant peak in MS spectra, it can be concluded that peak 3 in the 1H–15N HSQC spectra can be assigned to [Atox1–Pt(Ime)2]. The same adducts were formed in the reaction of trans-EE with CuI-Atox1, however, the intensity of a1 decreased after 1 h of reaction and a2 became dominant after 12 h of reaction (Fig. 4B). This result indicates that copper binding promotes the conversion of the Atox1 adduct (a1) to the Atox1– trans-EE–DTT ternary complex (a2). The formation of this ternary complex could be the preceding step of the release of trans-EE from Atox1 to DTT observed in the real-time NMR study. Fig. 5 shows the ESI-MS results of the reactions between Atox1 and trans-PtTz. Nearly identical adducts were generated in the reactions of apo- and CuI-Atox1. The adduct (b1) at m/z 997.10, which was assigned to [Atox1 + Pt(NH3)(Tz) + 6H]8+, is always dominant in both reactions. This composition should represent adduct 4 in the NMR spectra. Four minor adducts (b2–b5) were also detected. In all these adducts, two carrier ligands (NH3 and thiazole) remain coordinated to platinum; this result is also observed in the reaction of trans-EE. Consistent

Fig. 4 Time-dependent ESI-MS spectra of Atox1 in the reaction with trans-EE. (A) apo-Atox1; (B) CuI-Atox1. Peaks were assigned to [Atox1 + Pt(Ime)2 + 6H]8+ (a1, m/z 1002.51); [Atox1 + Pt(Ime)2 + DTT + 6H]8+ (a2, m/z 1021.75); [Atox1 + 2Pt(Ime)2 + 4H]8+ (a3, m/z 1045.02); [Atox1 + 2Pt(Ime)2 + DTT + 4H]8+ (a4, m/z 1064.35); [Atox1 + 2Pt(Ime)2 + 2DTT + 4H]8+ (a5, m/z 1083.67).

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Fig. 5 Time-dependent ESI-MS spectra of Atox1 in the reaction with equimolar trans-PtTz. (A) apo-Atox1; (B) CuI-Atox1. Peaks were assigned to [Atox1 + Pt(NH3)(Tz) + 6H]8+ (b1, m/z 997.10); [Atox1 + 2Pt(NH3)(Tz) + 4H]8+ (b2, m/z 1033.94); [Atox1 + 2Pt(NH3)(Tz) + DTT + 4H]8+ (b3, m/z 1053.34); [Atox1 + 3Pt(NH3)(Tz) + DTT + 2H]8+ (b4, m/z 1090.19); [Atox1 + Pt(NH3)(Tz) + DTT + 6H]8+ (b5, m/z 1016.22).

with the NMR results, the adduct b1 formed faster and also decreased more rapidly in the reaction of CuI-Atox1 than that of apo-Atox1, further supporting the conclusion that the copper coordination enhances the reaction rate of trans-PtTz with Atox1 and promotes the trans-platinum transfer from Atox1 to DTT. Platinum binding sites Previous studies show that cisplatin binds to the copper coordinating residues (Cys12 and Cys15) of Atox1 regardless of the presence of copper or not.15,22 Here, we analyzed the effect of copper coordination on the trans-platinum binding sites in Atox1 using tandem MS with trypsin digestion. After digestion, the same platinated peptides were detected in the reaction of apo-Atox1 as those in the reaction of CuI-Atox1 for each trans-platinum complex (Fig. 6 and Fig. S2, ESI†). Both trans-EE and trans-PtTz were found to bind to the same peptide fragment (H4EFSVDMTC12GGC15AEAVSR21) as cisplatin does. Additionally, trans-EE could also bind to the sequence of V40CIESEHSMDTLLATLK56, which contains a potential platinum binding site Cys41 (Fig. S2, ESI†). This additional binding site may relate to the multi-platinated Atox1 adducts. No corresponding fragment was detected in the adducts of cisplatin or trans-PtTz, probably due to the low abundance in the MS spectra. Tandem MS was used to identify the platinum binding sites by analyzing the fragmentation of the platinated peptide. For the reaction of trans-PtTz, the [P1 + Pt(NH3)(Tz) + H]3+ ion (m/z 731.71) was selected as the precursor ion for collision-induced dissociation experiments. The resulting ESI-MS/MS spectra and corresponding fragmentation schemes are shown in Fig. 7. The fragment ions (b and y) were annotated according to the Biemann nomenclature.23 On the other hand, the platinated fragments were identified by the characteristic isotope pattern of the platinum and annotated as b* and y*. In Fig. 7B, the largest b8 in the bn ions and the smallest b9* in the bn* ions indicate that C9 (Cys12 in Atox1) should be a platination site

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Species observed in the ESI-MS spectra

Pt agents

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Common species 8+

Formula

Observed m/z

Calculated m/z

trans-EE

a1: a2: a3: a4: a5:

[Atox1 [Atox1 [Atox1 [Atox1 [Atox1

+ + + + +

Pt(Ime)2 + 6H] Pt(Ime)2 + DTT + 6H]8+ 2Pt(Ime)2 + 4H]8+ 2Pt(Ime)2 + DTT + 4H]8+ 2Pt(Ime)2 + 2DTT + 4H]8+

C338H567N91O108S6Pt C342H577N91O110S8Pt C344H579N93O110S6Pt2 C348H589N93O112S8Pt2 C352H599N93O114S10Pt2

1002.51 1021.75 1045.02 1064.35 1083.67

1002.62 1021.87 1045.00 1064.26 1083.51

trans-PtTz

b1: b2: b3: b4: b5:

[Atox1 [Atox1 [Atox1 [Atox1 [Atox1

+ + + + +

Pt(NH3)(Tz) + 6H]8+ 2Pt(NH3)(Tz) + 4H]8+ 2Pt(NH3)(Tz) + DTT + 4H]8+ 3Pt(NH3)(Tz) + DTT + 2H]8+ Pt(NH3)(Tz) + DTT + 6H]8+

C335H559N91O106S7Pt C338H563N93O106S8Pt2 C342H573N93O108S10Pt2 C345H577N95O108S11Pt3 C339H569N91O108S9Pt

997.10 1033.94 1053.34 1090.19 1016.22

997.11 1033.98 1053.23 1090.11 1016.36

Fig. 6 Selected ESI-MS spectra of platinated peptides from trypsin digestion of (A) Atox1 and (B) CuI-Atox1 incubated with trans-PtTz for 4 h. The isotopic distribution of the f1 peak (the most abundant isotopomer at m/z 731.71, [P1 + Pt(NH3)(Tz) + H]3+), is well consistent with the simulated pattern (C). P1 represents the peptide H4EFSVDMTC12GGC15AEAVSR21.

and the other binding site is involved in the G10-R18 sequence. There is no y* ions so that the other site could not be directly read from the fragmentation result through the comparison of the yn ions with the yn* ions. However, the Pt2+ should favor bonding with sulfur ligands based on coordination chemistry. Therefore, the C12 (Cys15 in Atox1) is the most potential coordination site in the G10-R18 sequence. A similar result was obtained for the reaction of trans-PtTz with CuI-Atox1, indicating that trans-PtTz still binds to the copper coordinating residues in the presence of copper (Fig. 7D). The tandem MS experiments were also performed on the samples from the reaction of Atox1 with trans-EE, and results show that trans-EE binds to the copper binding sites in both apo-Atox1 and holo-Atox1 (Fig. S3, ESI†). Thus, copper coordination does not change the platinum binding sites, although it modulates the platination rate of Atox1 and the trans-platinum transfer from Atox1 to DTT.

Discussion Increasing evidence indicates that copper proteins are involved in the mechanism of platinum drugs.6,7,24,25 The copper

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Fig. 7 ESI-MS/MS spectra of the triply charged ion f1 at m/z 731.71 from trypsin digestion of (A) Atox1 and (C) CuI-Atox1 treated with trans-PtTz for 4 h. Fragmentation schemes based on the spectra (A) and (C) are shown in (B) and (D), respectively.

chaperone Atox1 could bind to cisplatin and has been proposed to be involved in the intracellular trafficking and drug resistance of cisplatin.6 Recently, we have found that copper binding could promote the interaction of Atox1 with cisplatin.15 In order to investigate the influence of the configurations of platinum complexes on the reactions of Atox1, we carried out detailed

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NMR and MS studies to probe the reactivity of Atox1 to two antitumor active trans-platinum complexes in the absence and presence of copper. Results reveal that trans-platinum complexes also preferentially bind to the copper coordinating residues (Cys12 and Cys15) in both apo-Atox1 and CuI-Atox1. However, copper coordination facilitates the platination of Atox1 by the trans-platinum complexes. Generally, metal binding could significantly lower the reactivity of metalloproteins towards platinum compounds, especially when the metal shares the same coordinating residues as the platinum.26,27 The results here indicate that the binding of copper and platinum to Atox1 is not simply competitive. It has been proposed that the Cu–Pt interaction is present in the reaction of CuI-Atox1 only with cis-platinum complexes containing two exchangeable leaving groups.13,28 In this work, however, we found that Cu(I) binding promotes the reactions of Atox1 also with the trans-platinum complexes. The structural investigations on Atox1 have demonstrated that, upon Cu(I) binding, both Cys12 and Cys15 show decreased dynamics and their side-chains become closer to each other.29,30 In addition, the more solvent exposure of Cys15 thiol in CuI-Atox1 could make the cysteines more accessible for platinum binding. Cisplatin has been reported to bind to Atox1 and lead to the protein unfolding and aggregation in vitro.13 Our previous result showed that in the reaction of cisplatin, the enhanced Atox1 platination by the copper coordination promotes the Atox1 aggregation.15 Here we demonstrated that the trans-platinum complexes also induced the aggregation of Atox1, and this aggregation is more obvious in the reaction of CuI-Atox1 (Fig. S4, ESI†). However, much less protein aggregation was observed in reactions of the trans-platinum complexes, even though they are more reactive to Atox1 than cisplatin. These divergences could be correlated with the different coordination chemistry of these platinum complexes. It has been observed that cisplatin could release the ammine ligands in the reaction with Atox1.15 The two additional coordination sites generated by loss of ammine ligands from cisplatin can facilitate the formation of inter-molecular complexes. In contrast, the nitrogen ligands are always retained in the reaction of trans-platinum complexes (Fig. 4 and 5). It has been proposed that the aggregation of Atox1 could associate with the cisplatin resistance.13 Results here highlight the different mechanisms of Atox1 in the cellular process of trans- and cis-Pt complexes; in addition, this process is also modulated by the copper binding. Kinetic properties have been proved to be crucial in the determination of the drug activity of platinum complexes. It is well known that higher reactivity of transplatin causes its lower antitumor activity than cisplatin.31,32 Replacing the ammine ligands of transplatin with bulky ligands could lead to lower reactivity but higher antitumor activity.4 For instance, trans-EE shows a similar DNA binding rate to cisplatin.21 Nevertheless, the two trans-platinum complexes are much more reactive than cisplatin in the reactions with both apo- and holo-Atox1. This result could be explained by the strong trans effect of the thiol group.33 With the binding of the first cysteine residue, the other leaving group is trans to the thiol group in the trans-platinum

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Metallomics

complexes, which makes the second substitution very fast. This is not the case in the reaction of DNA, in which the coordination of guanine N7 has much lower trans effect. On the other hand, it is interesting to note that DTT has different effects between the reactions of trans- and cis-platinum complexes. Cu(I)-binding facilitates the DTT-induced trans-platinum release from Atox1 but suppresses the cisplatin release, although Cu(I)-binding promotes the platination of Atox1 by all three platinum complexes. The significantly different reactivity and binding stability of platinum complexes to proteins could correlate to the diverse mechanisms of various drugs. As a copper chaperone protein, Atox1 executes its function by binding to cuprous ions and delivering them to the target proteins (ATP7A and ATP7B) in cells.34,35 ATP7A and ATP7B have been proved to be involved in the resistance of platinum drugs.36,37 Both ATP7A and ATP7B contain six metal binding domains (MBDs) in the cytoplasmic N-terminal domain, and the overall structure and the conserved copper binding motif of each MBD are very similar to that of Atox1.38 Therefore, it is very likely that the copper binding may also promote the platination of ATP7A and ATP7B, which consequentially influences the sequestration or efflux of platinum complexes. Moreover, more folded trans-platinum–Atox1 species and immediate trans-platinum release from Atox1 suggest a possible mechanism of copper in the regulation of the trafficking of trans-platinum complexes: copper binding promotes the transfer of trans-platinum complexes from Atox1 to ATP7A and ATP7B, for the subsequent cellular export.

Conclusions In conclusion, this work demonstrates that copper binding promotes the platination of Atox1 by two antitumor active trans-platinum complexes, although platinum binds to the copper coordinating residues of the protein. In comparison to cisplatin, trans-platinum complexes are much more reactive to Atox1, however, induce less protein aggregations. In addition, cuprous ions demonstrate different effects on the platinated Atox1 adducts of trans-platinum relative to cisplatin. The CuI-coordination facilitates the trans-platinum transfer from Atox1 to DTT, whereas significantly suppresses the platination of DTT by cisplatin. As Atox1 is a copper chaperone and executes functions with copper coordination, the modulation of platination by copper indicates that the Atox1 protein plays different roles of cis- and trans-platinum agents in the intracellular trafficking, and could be correlated with cross resistance between copper and platinum drugs.

Acknowledgements This work was supported by the National Basic Research Program of China (973 Program, 2012CB932502), the Chinese National High-Tech Research Grant (2006AA02A321), the National Science Foundation of China (U1332210, 21171156, 21135006, 21020102039), the Ministry of Education of China

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(SRFDP 20133402110041) and the Fundamental Research Funds for the Central Universities.

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Metallomics, 2014, 6, 491--497 | 497

Copper binding modulates the platination of human copper chaperone Atox1 by antitumor trans-platinum complexes.

The transport system of platinum-based anticancer agents is crucial for drug sensitivity. Increasing evidence indicates that the copper transport syst...
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